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Development and Validation of

Analytical Methods for Elemental Sulphur in Alberta Soils

June 2015

Jun 2015 Development and Validation of Analytical Methods for Elemental Sulphur in Alberta Soils

© 2015 Government of Alberta

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ISBN 978-1-4601-2146-7 (Print) ISBN 978-1-4601-2147-4 (PDF) Website: Directive for Monitoring the Impact of Sulphur Dust on Soils

Development and Validation of Analytical Methods For Elemental Sulphur in Alberta Soils

Disclaimer: Any mention of trade names of commercial products does not constitute an endorsement or recommendation for use.

Citation: This document should be cited as: Alberta Environment and Parks, 2015. Development and Validation of Analytical Methods for Elemental Sulphur in Alberta Soils, Prepared by Maxxam Analytics International Corporation for Alberta Environment and Parks, June, 2015, Edmonton, Alberta.

Any comments, questions or suggestions on this document may be directed to: Land Policy Branch Alberta Environment and Parks Oxbridge Place, 10th Floor 9820 – 106th Street Edmonton, Alberta T5K 2J6 Tel: 780-415-2585 Fax: 780-422-4192 Email: [email protected]

Additional copies of this document may be obtained by contacting: Information Centre Alberta Environment and Parks Main floor, Great West Life Building 9920 - 108 Street Edmonton, Alberta T5K 2M4 Email: [email protected] Website: aep.alberta.ca

© 2015 Government of Alberta Copyright of this publication, regardless of format, belongs to Her Majesty the Queen in right of the Province of Alberta. Reproduction of this publication, in part or whole, regardless of purposes, requires the prior written permission of the Alberta Environment and Parks.

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Development and Validation of Analytical Methods for Elemental Sulphur in Alberta Soils

Developed by Maxxam Analytics

for

Alberta Environment and Parks

June 2015



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Table of Contents

1 Background ......................................................................................................................... 5 2 Initial Investigations ........................................................................................................... 5

2.1 Standard Integrity................................................................................................... 5 2.2 Evaluation of Extraction Solvents ........................................................................... 7

2.2.1 Solubility .................................................................................................... 7 2.2.2 Extraction Efficiency .................................................................................. 8

2.3 Evaluation of Instrument Response, Columns and Mobile Phases ...................... 10 2.4 Instrument Detection Limits ................................................................................ 16 2.5 Initial Instrument Conditions................................................................................ 16

2.5.1 Initial Conclusions .................................................................................... 17 2.6 Sensitivity and Freedom from Interferences ....................................................... 17

2.6.1 Sample Extraction.................................................................................... 17 2.6.2 Colourimetric Analysis and Interference ................................................. 21 2.6.3 HPLC Parameter Adjustments ................................................................. 22 2.6.4 Sample Dilution and Analysis .................................................................. 24 2.6.5 Robustness .............................................................................................. 27

2.7 Selection of Protocol for Validation ..................................................................... 28 3 Development of The Final Optimized Analytical Method and Recommended

Operating Protocols .......................................................................................................... 28 3.1 HPLC Method Optimization .................................................................................. 28

3.1.1 Optimization of the Instrumental Response ........................................... 28 3.1.2 Optimization of the Mobile Phase .......................................................... 28 3.1.3 Instrument Parameters ........................................................................... 34

3.2 Method Validation ............................................................................................... 35 3.2.1 Calibration of Linear Range ..................................................................... 35 3.2.2 Method Working Range .......................................................................... 36 3.2.3 Method Detection Limits (MDL) .............................................................. 37 3.2.4 Method Blank .......................................................................................... 38 3.2.5 Precision and Accuracy, Robustness ....................................................... 38 3.2.6 Selectivity ................................................................................................ 39 3.2.7 Second Source Verification ..................................................................... 39 3.2.8 Certified Reference Material (CRM) ........................................................ 40

4 Summary ........................................................................................................................... 41 5 References......................................................................................................................... 42 6 Appendices ........................................................................................................................ 42 7 Attachments ...................................................................................................................... 42 8 Acknowledgements .......................................................................................................... 43



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1 Background

Deposition of air-borne sulphur dust from industrial facilities that handle or process solid

elemental sulphur has the potential to cause soil acidification at or near those sites in Alberta.

Lack of suitable analytical methods for elemental sulphur (S8) has become a challenge for soil

monitoring for industry operators and regulators. An acetone extraction-sodium cyanide

colorimetric analysis method (Maynard and Addison, 1985) has been used in Alberta but

environmental consultants and laboratories complained about the high detection limit and

questioned its reliability. A chloroform extraction-HPLC analysis method developed in New

Zealand (Watkinson et al., 1987) was reported to work well with a wide range of agricultural

mineral soils and some sediments. It was not clear if it would be suitable in Alberta because the

impact of elemental sulphur on soils is frequently monitored in forested sites where organic

materials from forest litter may interfere. There was also a need to assess if the above-noted

methods were compatible with the analytical laboratories in Alberta. As chloroform is a toxic

substance, alternative solvents of similar extracting capacity for elemental sulphur needed to be

explored.

Maxxam Analytics was previously retained by the former Department of Alberta Environment

and Sustainable Resource Development (ESRD) to calibrate and refine the above-noted methods

and, if needed, to develop alternative analytical methods and subsequent standard operating

procedures (SOP). This project was conducted with practical input from a network of

commercial laboratories in Alberta. Maxxam Analytics also coordinated a round robin project,

with participating members from the network of commercial laboratories in Alberta. A final

project report was submitted to ESRD in May and the round robin report in July of 2013.

This combined report is prepared at the request of Alberta Environment and Parks (AEP) for

operational applications and has incorporated elements of the two foregoing documents. The

rationale, approaches, and results presented in this report form the basis for the SOP, which

promotes consistency in implementation of the analytical method. The SOP is included as

Attachment A and the round robin results as Attachment B.

2 Initial Investigations

Preliminary experiments were conducted to assess sample preparation and analysis options.

Once completed, the data were compiled and reviewed with ESRD in order to select the

optimum procedure for full method validation.

2.1 Standard Integrity

Literature references and our initial investigations showed that elemental sulphur working

standards contained some S6 and S7 and perhaps polymeric species, as well as S8.

Representative chromatograms of a 200 µg/mL injection of the reference standard are shown in

Figures 1a and 1b. Our chromatographic investigations showed that for the primary and



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secondary standards employed in this study, the maximum area of these species combined was



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Figure 1b. Chromatogram of 200 µg/mL injection, C18 column (high capacity) - Expanded Y-

axis

Table 1. Summary of Impurities in Sigma-Aldrich S8 Standard

id µg/ml 0.87 min % of S8 1.0 min % of S8 1.4 min % of S8 2.4 min % of S8 3.4 min % of S8 4.1 min 5.2 min (S8)

std 2 0.4 0.58 743.6% 0.08

std 3 2 0.54 154.3% 0.35

std 4 4 0.35 47.3% 0.74

std 5 10 0.49 19.9% 2.46

std 6 20 0.21 5.1% 4.10

std 7 40 0.3 3.4% 0.02 0.23% 0.02 0.23% 0.075 0.86% 8.70

std 8 100 0.23 1.0% 0.038 0.17% 0.038 0.17% 0.22 0.99% 22.19

std 9 200 0.32 0.7% 0.083 0.18% 0.013 0.03% 0.083 0.18% 0.51 1.14% 0.015 44.88

Summary of Peaks and Peak Areas for Standard S8 (10 µL injected)

2.2 Evaluation of Extraction Solvents

2.2.1 Solubility

The first step was to determine the solubility of S8 in the various solvents. Three replicates were

prepared in each of acetone, dichloromethane (DCM) and methanol (MeOH); one replicate was

prepared in chloroform (CHCl3) (Table 2). Excess S8 was mixed with solvent, sonicated for one

hour, centrifuged and decanted. The supernatant was analyzed colourimetrically.



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Table 2. Summary of Elemental Sulphur Solubility in Acetone, DCM, Chloroform, Methanol

Solvent Solubility (µg/mL)

Comments

Acetone 604, 623, 610 Incomplete dissolution, very small amount of dusty sediment

Dichloromethane 6185, 6089, 6720 Incomplete dissolution, visible amount of sediment

Chloroform 6670 Incomplete dissolution, visible amount of sediment

Methanol 260, 247, 265 Incomplete dissolution, visible amount of dusty sediment

Acetone, DCM and chloroform are extraction solvents; methanol is used as the mobile phase. As

can be seen, the solubility of S8 in chloroform and DCM are the same at approximately

6,500 µg/mL. Solubility in acetone is about an order of magnitude less at 600 µg/mL.

2.2.2 Extraction Efficiency

Extraction efficiency was evaluated by preparing high concentration spikes of a clay matrix (40%

clay by hydrometer) containing 9% and 2% total organic carbon, respectively.

All samples were dried at 55 ± 5°C to eliminate moisture. This minimizes microbial influence on

sample integrity and also reduces problems when extracting with hydrophobic solvents. The

samples were then ground to < 2 mm to provide sample homogeneity.

Individual 2 g aliquots were spiked with a concentrated S8 standard in DCM. Spiked samples

were mixed and the DCM allowed to evaporate overnight in the fumehood. Extraction solvent,

20 mL, was added and the samples sonicated for 30 minutes, followed by tumbling in a rotary

mixer for one hour. Samples were extracted at 5:1 and 10:1 solvent:soil (volume:weight) ratios.

Extracts were centrifuged, decanted and analyzed both colourimetrically and by HPLC.

Recoveries using both analytical techniques were similar. Graphs of the results are displayed in

Figures 2a and 2b, below.



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Figure 2a. Acetone Extraction Efficiency

Acetone extraction efficiency

0.0

20.0

40.0

60.0

80.0

100.0

120.0

140.0

160.0

0 100 200 300 400 500 600 700 800

Conc. mg/L

% r

eco

very

Figure 2b. Chloroform/Dichloromethane Extraction Efficiency

DCM extraction Efficiency

60.0

70.0

80.0

90.0

100.0

110.0

120.0

0 1000 2000 3000 4000 5000 6000

conc. mg/L

% r

ec

ov

ery

The data show that extraction efficiency is dependent on the solubility of S8 in the various

solvents. Again, data for chloroform and DCM were similar showing 100% efficiency at spike

extract concentrations up to 6,000 mg/L (extract concentration). Acetone shows excellent

recoveries up to at least 400 mg/L. Going forward, depending on the solvent, if analysis yields

values at these levels or above, re-extraction using a larger solvent:soil ratio would be required.

CHCl3 / DCM Extraction Efficiency



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2.3 Evaluation of Instrument Response, Columns and Mobile Phases

Two systems were evaluated: 1) the Watkinson et al., 1987 reference method, which employs a

polymeric reversed phase (PRP) column and adsorption (solid/liquid) chromatography with a

chloroform-methanol mobile phase, and 2) the most commonly used analytical system, a C18

column with partition chromatography using methanol-water as the mobile phase (Azarova et

al., 2001).

From scans of standards, it was determined that the highest molar absorptivity achievable for S8

occurs at 220 nm. Because the UV cutoff for chloroform is 240 nm, it was necessary to use the

less sensitive 254 nm peak for the PRP work. The chromatograms below (Figures 3a to 3c) show

the relative response of the same standard at various wavelengths using the C18 column.



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Figure 3a. S8 Response at 220 nm



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Figure 3b. S8 Response at 254 nm



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Figure 3c. S8 Response at 280 nm

Analysis using a chloroform mobile phase and polymeric column was plagued with difficulty. The

column is a specialty item and took several weeks to obtain. The chloroform attacked the pump

seals leaving the system not functional until the Teflon® seals were obtained and installed (two

weeks). Limited data showed adequate separation of the S8 peaks and adequate sensitivity.



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However, since the mandate was to develop a method that could be easily adopted by other

commercial labs and, more importantly, conventional partition chromatography using a

standard C18 column showed equal or better performance, efforts focused on the development

of that method.

Example chromatograms (Figures 3d to 3h) from the two methods show the improved

chromatography using C18 methanol-water separation relative to the PRP chloroform-methanol

system.

Figure 3d. C18 Method – High Organic Clay in Acetone (2 µg/mL)

Figure 3e. PRP Method – High Organic Clay in Acetone (2 µg/mL)



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Figure 3f. PRP Method – Lodgepole Pine Litter in Acetone (10 g/mL)

Figure 3g. C18 Method – Lodgepole Pine Litter in Acetone (10 µg/mL)



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Figure 3h. C18 Method – White Spruce Litter in Acetone (10 µg/mL)

2.4 Instrument Detection Limits

Instrument detection limits (IDL) were estimated by replicate injections of a low level standard

or a 3:1 signal to noise ratio (10 µL injected).

• IDL PRP / CHCl3, 0.4 µg/mL

• IDL C18 / 90:10 MeOH:H2O, 0.08 µg/mL

Note that these are IDLs and actual MDLs determined when replicates are carried through the

entire analytical process will be higher. Nonetheless, the five-fold improvement in sensitivity

using the C18 column and detection at 220 nm was very encouraging.

2.5 Initial Instrument Conditions

For method optimization, we targeted isocratic elution conditions that provided a capacity

factor (retention factor, k’) about 5.2 This was selected after reviewing the capacity factors

employed by Watkinson et al., for soil extraction (k’ = 5.2), and Azarova et al., for sediment

extraction (k’ = 5.4).

For the PRP method, a Hamilton PRP-1 column was selected (4.6 x 15 mm, 5 µm particle size).

The 4.1 x 150 mm, 10 µm particle size column specified by Watkinson et al. was not available.

2 Capacity factor: k’ = (tR – tM)/tM



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Isocratic elution with 1:1 CHCl3:MeOH (1.0 mL/min) yielded k’ = 2.9 and retention time (tR)= 5.9

min. Given that the S8 peak was asymmetrical and very broad (likely due to the solid/liquid

interaction energetics) and the S8 peak was baseline-separated from the interference in the

worst-case leaf-litter sample, conditions providing a larger k’ were not investigated.

For the C18 method, initially a Waters X-Bridge C18 column was employed (3.0 x 150 mm, 3.5

µm particle size). Isocratic elution conditions with 90:10 MeOH:H2O at 0.5 mL/min provided k’ =

2.9 and tR = 6.2 min with excellent peak shape. For enhanced capacity and resolution (and

reduced column cost) an AkzoNobel Kromasil® C18 column (4.6 x 100 mm, 3.5 µm particle size)

was employed later. Isocratic elution with 90:10 MeOH:H2O at 2.3 mL/min provided k’ = 7.7 and

tR = 5.2 min. Again peak shape was acceptable, but such high values of k’ are typically not

preferred due to their negative impact on peak resolution. Increasing eluent strength to 95:5

MeOH:H2O while maintaining flow of 2.3 mL/min provided k’ = 4.4 and tR = 2.7 min. Under these

conditions, there was some co-elution with the tail of polar interferences from pine and spruce

leaf litters. Further refinements in elution strength will be undertaken to optimize retention and

separation from co-extracted interferences in the final method validation. It may be necessary

to return to a 15 cm column if the desire is to have baseline separation between interferences

and S8 in all cases.

2.5.1 Initial Conclusions

• For S8 dissolved in solvent, detection at 220 nm with methanol-water mobile phase

provides the best sensitivity. (Note: UV cutoffs for methanol, acetone, DCM and

chloroform are, respectively: 205, 330, 230, and 240 nm.)

• We cannot add acetone to the C18 mobile phase to increase S8 solubility because of

the 330 nm UV cutoff.

• For the PRP method, we will lose about 50% of sensitivity by having to detect at the

higher wavelengths.

2.6 Sensitivity and Freedom from Interferences

2.6.1 Sample Extraction

Sensitivity and freedom from interferences were evaluated by low level spikes of the high and

low organic clays and three leaf litter samples provided by ESRD. The following approaches were

used:

• Two clay samples and three leaf litter samples extracted with three solvents at 5:1

and 10:1 solvent:soil ratios,

• 2 g soil and 10 or 20 mL solvent used,

• Spiked at 20 and 100 µg/g S8 in the soil, corresponding to 2 to 10 µg/mL S8 in the

10:1 extract and 4 and 20 µg/mL S8 in the 5:1 extract.



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All samples were extracted and centrifuged as described in Section 2.2.2. The leaf litter samples

proved more challenging, particularly for the DCM and chloroform extracts. Because of the

density of these solvents, significant floating and suspended materials remained after

centrifuging. These samples had to be centrifuged a second time and some were filtered before

analysis. Also, the 5:1 extracts were more difficult to separate than the 10:1. In contrast, the

acetone extracts settled readily and could be decanted after a single centrifugation.

The leaf litter extracts were all highly coloured. The high and low organic clay samples were

colourless. Example chromatograms of unspiked 10:1 acetone extracts using an expanded y-axis

scale show little interference in the S8 elution range, indicated by the red marker (Figures 4a to

4e). Since acetone extracts were run undiluted, these represent the worst-case scenario. As

expected, the leaf litter samples displayed significant interferences, but these mostly elute prior

to S8. Therefore, the mobile phase is capable of separating the signals associated with the

interference from those of S8. A chromatogram of 20 µg/mL S8 standard in acetone is presented

for comparison (Figure 4f).

Figure 4a. High Organic Clay



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Figure 4b. Clay

Figure 4c. White Spruce Leaf Litter



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Figure 4d. Lodgepole Pine Leaf Litter

Figure 4e. Aspen Leaf Litter



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Figure 4f. S8 Standard in Acetone (20 µg/mL)

All three solvent extracts were analyzed by HPLC. Acetone extracts were also analyzed

colourimetrically. The data are tabulated in the Master Data Table in Appendix 1.

2.6.2 Colourimetric Analysis and Interference

The colourimetric analyses showed good recoveries for the clay samples, averaging 107 ± 14 %.

Note that the unspiked clay samples gave small positive readings (0.5 µg/mL) by the

colourimetric method. HPLC analysis confirmed a similar level (0.9 µg/mL) of S8 in the high

organic clay sample (9% organic clay) but not in the low organic clay sample (2% organic clay)

(see Figure 5). Taking this into account, the average recovery was 97%.

As expected, colourimetric analysis of the leaf litter extracts showed significant background

levels, ranging from 2.4 to 7.7 µg/mL. These levels were not confirmed by HPLC, thus this is a

true background interference, resulting in high and erratic recoveries in the spikes.

Attempts were made to correct the data by background subtraction, but this did not improve

the recoveries. Thus, colourimetric analysis is not suitable for coloured extracts.



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Figure 5. Sulphur Recovery in Unspiked High Organic (9%) and Low Organic (2%) Clay

Samples

2.6.3 HPLC Parameter Adjustments

A 90:10 MeOH:H2O mobile phase with a 15 cm C18 column provided clear separation of the S8

peak from all interferences while maintaining a reasonably short run time of < 12 minutes

(Figures 6a to 6c).



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Figure 6a. White Spruce Leaf Litter Blank Soil, Extract Spiked

Figure 6b. Lodgepole Pine Leaf Litter Blank Soil, Extract Spiked



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Figure 6c. Aspen Leaf Litter Blank Soil, Extract Spiked

The linear range will be limited by the solubility of S8 in the mobile phase. In the initial

investigations, linearity has been established up to 200 µg/mL using the standard 10 µL extract

injection. The upper limit of the linear range will be established during validation.

Using these protocols, and assuming a 10:1 solvent:soil ratio, the working range for the acetone

extracts is estimated to be 2 – 3,000 µg/g S8 and 20 to 30,000 µg/g (3%) S8 for DCM extracts.3

This assumes linearity can be extended to 300 µg/mL in the extracts. Early indications are that

linearity may extend to 500 µg/mL, which is near the solubility limit of S8 in acetone.

2.6.4 Sample Dilution and Analysis

Acetone extracts were run undiluted. DCM and chloroform extracts were diluted 10x with

acetone prior to injection to ensure miscibility of the chlorinated solvents in the mobile phase.

Note that the 9 or 10 µL of acetone injected is insufficient to interfere with the S8 peak. In the

example chromatogram below (Figure 7) the large peak at 1.6 min. is due to acetone.

3 DCM (and chloroform) extracts are diluted 10x in either methanol or acetone prior to injection. Neat

DCM and chloroform are not miscible with the methanol-water mobile phase. We observed no miscibility problems during chromatography with the chlorinated solvent extracts when diluted as described. Dilution in acetone provides a means to accommodate the solvent with higher S8 solubility and so is preferred for higher concentration extracts. We recommend acetone as diluent going forward.



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Figure 7. S8 Standard in Acetone (20 µg/mL)

All three extraction methods provided good recoveries. Table 3 gives the averages and standard

deviations for the recoveries of the five samples using three different solvents.

Table 3. Average Sulphur Recovery - All Solutions (DCM, Acetone, Chloroform)

Extract Ratio (Solvent to

Soil)

Design µg/mL

Average Recovery

Standard Deviation

10 to1 2 130% (106%) 26%

5 to 1 4 104% 23%

10 to 1 10 88% 11%

5 to 1 20 87% 12%

The chloroform extracts exhibited a high bias on the 2 µg/mL extracts, which may be an artifact

since these were analyzed some time after the rest. Excluding these values, the mean recovery

was 106%.

The larger variability observed for the most dilute extracts, 2 µg/mL and 4 µg/mL, is in keeping

with samples having concentrations near the detection limit. A chromatogram of standards at

0.4 µg/mL (std 1), 1.0 µg/mL (std 2) and 2.0 µg/mL (std 3) is shown in Figure 8a. Example

chromatograms of 2 µg/mL extracts are shown in Figures 8b to 8d below.



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Figure 8a. Standards at 0.4, 1.0, 2.0 µg/mL

Figure 8b. High Organic Clay Spiked, Extract at 2 µg/mL

Figure 8c. Lodgepole Pine Leaf Litter Spiked, Extract at 2 µg/mL



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Figure 8d. White Spruce Leaf Litter Spiked, Extract at 2 µg/mL

The average recoveries for all extracts determined using each of the tested solvents (excluding

the 2 µg/mL chloroform extracts) are shown in Table 4.

Table 4. Average Recoveries in Acetone, DCM and Chloroform

Solvent Average Recovery

Number of Replicates

Acetone 98% 12

DCM 103% 12

Chloroform 101%* 8

*excluding 2 µg/mL extracts

2.6.5 Robustness

Relatively rapid clogging of the column and concomitant backpressure build-up was observed.

This was probably due to pine pitch from the leaf litter extracts, not particulate matter in the

extracts, as a 2 µm in-line filter was employed immediately upstream of the column and extracts

were filtered prior to injection. In response, various guard columns and column cleaning regimes

were investigated. The optimum guard column and cleaning protocol will be included in the final

SOP. At this point it appears that the lodgepole pine extracts cause the worst column

contamination. Cleaning with polar solvents (acetonitrile, acetone, methanol) either on their

own or in various combinations with water is not sufficient to eliminate the contamination.

Isopropanol, hexane and/or heptane may be moderately effective. Investigations were

conducted to identify a guard column that is most effective in trapping the interference in order

to avoid having to implement either a periodic column-cleaning regimen or gradient elution with

a suitable solvent to elute the interference at the end of each run.



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2.7 Selection of Protocol for Validation

The preceding data was presented to ESRD, a protocol selected for optimization, and, after

optimization, the method was documented in a standard operating procedure. The reasoning

for the protocol selection is detailed in Section 3 below.

3 Development of the Final Optimized Analytical Method and Recommended Operating Protocols

Based on the findings of the initial investigations and consultation with ESRD, method

optimization and validation were conducted on the following methodology:

• Extraction with acetone and DCM using 2 g soil and 20 mL solvent. Analysis using

reversed phase partition HPLC with a conventional C18 column, methanol-water

mobile phase and UV detection at 220 nm.

The reasoning for these choices is as follows:

• Government of Alberta requires a high level method for sites heavily impacted by

elemental sulphur and a low level method for remote areas less affected by longer

range aerial transport. Thus, two solvents were validated for use: DCM for high

levels because of the much greater solubility of S8, and acetone for low levels

because the extract can be run undiluted.

• DCM was chosen over chloroform because it provides equal performance to

chloroform, is a common laboratory solvent and is not a carcinogen.

• A 10:1 solvent:soil ratio was adopted as the optimum compromise between

providing adequate sensitivity and effective extraction and separation of the extract

from the solid matrix.

• The conventional C18 column was chosen instead of the PRP because it is widely

used in the laboratory community and provides superior sensitivity to the PRP.

3.1 HPLC Method Optimization

3.1.1 Optimization of the Instrumental Response

Selection of the 220 nm wavelength was discussed in Sections 2.3 to 2.4. Optimization of other

instrumental parameters is discussed below.

3.1.2 Optimization of the Mobile Phase

The most difficult sample matrix was lodgepole pine leaf litter. Although the original work

provided reasonable separation of the S8 peak from interferences, additional work was done to

improve the separation. Optimization included varying the methanol to water ratio to further

increase the capacity factor while maintaining a reasonably short run time. The optimized



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system is a compromise between separation and a realistic run time. The chromatogram at

normal scale expansion shows virtual complete separation from the interference. Expanded

scale shows that < 1% of the interference remains at the S8 elution time but that a peak of a

very low level standard is easily established.

Selectivity Evaluation

• Mobile phase 95:5 MeOH:H2O, k’ = 4.4

• Mobile phase 90:10 MeOH:H2O, k’ = 7.4

We found the worst-case scenario for selectivity occurs for acetone extraction/injection of

lodgepole pine soil samples. The acetone extracts were injected undiluted so a significantly

higher amount of co-extracted interference was injected relative to the DCM extracts diluted

10x prior to injection. Of the soils tested, lodgepole pine has the most challenging interferences.

These are, however, primarily polar and as such as they elute early. Our goal was to get

sufficient separation of the S8 peak from the polar interferences that elute starting immediately

after the dead volume (DV) time. When the column is run at 95:5 MeOH:H2O mobile phase, the

S8 elutes with an acceptable peak shape and capacity factor (4.4) but is still in the tail of the

polar interferences eluting prior, and the baseline is elevated by about 3.5 mAU relative to the

start of the run (Figure 9a). At 90:10 MeOH:H2O mobile phase, the S8 has separated further

from the polar interference, with a baseline elevation above the injection baseline of about 2.3

mAU (Figure 9b). The capacity factor here is now 7.4, which is considered quite high for an

elution of just one compound, and we see there is observable peak broadening due to the

higher column residence time. For this reason, an even longer elution time is not preferred.

We noted from the full-scale chromatograms that the DV peak (containing much of the polar

interference as well as the injection solvent, acetone) has a height of about 650 mAU.

Therefore, it may be considered that under either of the mobile phase running conditions, at the

time the S8 peak elutes the absorbance from the interference peak is >99% returned to baseline

(Figures 9c and 9d). From the chromatograms with the y-axis scaled to 200 mAU, which is more

representative of the full extent of the interference peak, it is more apparent that the S8 peak is

not significantly impacted by the interference peak. The peak separation and resolution are

better demonstrated with samples spiked at 100 µg/mL S8 (Figures 10a and 10b).

To achieve 100% return to baseline would require either a very long residence time for S8,

resulting in unacceptable peak broadening, or a 15 cm column, which would result in an

approximate 50% increase in retention time, although with less peak broadening than would be

seen on the 10 cm column, and a comparable increase in cycle time.

We believe these elution conditions are an appropriate compromise between adequate

separation from the interference peak, reasonable cycle time (sample throughput) and

adequate accuracy and precision of the S8 peak.



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Acetone Extracts

Figure 9a. HPLC chromatogram for the lodgepole pine leaf litter spiked at 20 µg/g and

extracted with acetone. Extract concentration 2 µg/mL S8, mobile phase 95:5

MeOH:H2O. Note: expanded y-axis, peak area 0.248.

Figure 9b. HPLC chromatogram for the lodgepole pine leaf litter spiked at 20 µg/g and


MeOH:H2O. Note the improved separation for S8 peak, expanded y-axis, peak area

0.280.



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Figure 9c. HPLC chromatogram for the lodgepole pine leaf litter spiked at 20 µg/g and


MeOH:H2O. Note: y-axis rescaled to 200 mAU, peak area 0.280.

Figure 9d. HPLC chromatogram for the lodgepole pine leaf litter spiked at 20 µg/g and


MeOH:H2O. Note: y-axis full scale at 700 mAU, peak area 0.280.



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Figure 10a. HPLC chromatogram for the lodgepole pine leaf litter spiked at 1,000 µg/g S8.

Extract concentration 100 µg/mL S8, mobile phase 90:10 MeOH:H2O. Note: y-axis

rescaled to 190 mAU.

Figure 10b. HPLC chromatogram for the lodgepole pine litter spiked at 1,000 µg/g S8. Extract

concentration 100 µg/mL S8, mobile phase 90:10 MeOH:H2O. Note: y-axis full scale

at 700 mAU.



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DCM Extracts

Initial investigations determined the solubility of elemental sulphur in DCM to be about ten

times greater than in acetone. DCM extracts were diluted 10x with acetone before HPLC analysis

in order to prevent damage to the needle seats and extend the linear range of the method by an

order of magnitude. Interestingly, the dilution procedure reduces the impact of the

interference, as shown in the chromatograms below (Figures 11a and 11b) where an equivalent

amount of S8 was injected, and S8 peak areas here and for the 2 µg/mL acetone injection

(Figures 9b to 9d) are equivalent.

Figure 11a. HPLC chromatogram for lodgepole pine leaf litter spiked at 200 µg/g, DCM extract

concentration 20 µg/mL S8, 2 µg/mL injected, mobile phase 90:10 MeOH:H2O, peak

area 0.268.



Page 34 of 78

Figure 11b. HPLC chromatogram for the DCM extract from lodgepole pine leaf litter spiked at

10,000 µg/g, extract concentration 1,000 µg/mL S8, 100 µg/mL injected, mobile

phase 90:10 MeOH:H2O.

3.1.3 Instrument Parameters

The final instrument conditions were:

• Column: C18, 100 mm x 4.6 mm ID, 3.5 µm particle size, or equivalent;

• Guard column: C 18, 10 mm x 4.6 mm ID, 5 µm particle size, or equivalent

(recommended for leaf litter soils);

• Column flow: 2.3 mL/min;

• Mobile phase: 90:10 MeOH:H2O (v:v);

• Wavelength: 220 nm;

• Column temp.: 45°C; and

• Injection volume: 10 µL.

Acetone extracts are injected neat. DCM extracts are injected after 10x dilution with acetone.

Note 1: Diluting DCM extracts less than 10x is not recommended unless a Teflon® needle seat is

employed. We observed near immediate deterioration of a standard needle seat with a

5x dilution of DCM extracts.

Note 2: Column contamination due to interferences from the lodgepole pine leaf litter extracts

was a significant problem. We finally determined that a C18 guard cartridge is the

simplest solution to this problem. If a high proportion of leaf litter samples is analysed,



Page 35 of 78

the guard cartridge must be replaced every 20-100 injections (i.e., daily). For minor

contamination that still makes it past the guard cartridge, cleaning the analytical column

with acetonitrile is sufficient. We sourced an inexpensive cartridge and holder for this

purpose.

3.2 Method Validation

Government of Alberta requires a method that has a low detection limit to monitor levels in

background and slightly impacted soils, as well as an alternative method for determining high

levels of elemental sulphur in heavily contaminated sites and elemental sulphur-containing

waste materials. Thus, two method validations are included. An acetone extract is proposed for

the former soils because the extract can be run undiluted on the HPLC, and a DCM extract for

the latter due to a much higher S8 solubility.

Method Validation includes:

• Calibration and linear range;

• Method working range;

• Method detection limit;

• Method blank;

• Precision and accuracy;

• Selectivity;

• Robustness;

• Second source verification; and

• Certified reference material (CRM) analysis.

3.2.1 Calibration of Linear Range

Experiments showed that a linear calibration equation, y = mx + b with 1/x weighting, provides

more accurate data at the low end of the curve. The calibration curve shows good linearity up to

400 µg/mL in the extract as injected (Figure 12). Using routine 2 g dried and ground sample and

20 mL solvent, the linear range can accommodate soils with elemental sulphur concentrations of

up to 4,000 µg/g with the acetone extraction and 40,000 µg/g (or 4%) with the DCM extraction

(including the 10x extract dilution).

Note: Acetone has significant volatility; therefore, good seals are required on autosampler

vials for both calibration standards and extracts. Acetone was selected for calibration

standard preparation due to the significantly better solubility of S8 in acetone relative to

methanol and acetonitrile. The chlorinated solvents, in which S8 has high solubility, are

not compatible with the mobile phase or standard reversed phase system seals because

they attack the standard seals of the HPLC pump.



Page 36 of 78

Figure 12. Linear Range of the HPLC Method

3.2.2 Method Working Range

The initial investigations of elemental sulphur solubility plus high level spikes in the method

validation showed effective extraction up to 400 µg/mL in an acetone extract and 6,500 µg/mL

in a DCM extract (assuming a 10:1 solvent:soil ratio). Any extracts with values above these levels

must be re-extracted with a higher solvent:soil ratio. Also, any extracts with concentrations

above the linear range of 400 µg/mL must be diluted into range. 400 µg/mL corresponds to

4,000 µg/g in a soil sample extracted with acetone, and 40,000 µg/g in a soil sample extracted

with DCM as a 10× dilution step for DCM extracts is incorporated in the protocol. The recoveries

for spikes at 90% of the linear range were between 88 and 108%. The values are shown in

Figure 13 below.



Page 37 of 78

Figure 13. Recovery of added elemental sulphur by the acetone-HPLC, and DCM-HPLC

methods. (Note: HOC is high organic clay, and LPP is lodgepole pine leaf litter; 360

and 3,600 are the concentrations in the extract in µg/mL.)

3.2.3 Method Detection Limits (MDL)

Method detection limits are determined as per EPA 40 CFR Part 136 Appendix B and CCME4 by

the analysis of low level spiked samples carried through the entire analytical process. The MDL is

defined as the standard deviation of the low level replicates multiplied by the t statistic for a

one-tailed test at a 0.01 confidence level for n-1 degrees of freedom (t = 2.998 for n = 8). The

spiking level must be between 1 and 10 times the calculated MDL. The MDL is intended to

restrict the chance of false positives to 1% when there is no analyte present in the sample.

Eight 2 g replicates were extracted with 20 mL of solvent. DCM extracts were diluted an

additional 10x prior to analysis. The results are presented in Table 5. Note that MDLs are

normally determined in a single run using a clean matrix such as Ottawa sand. By using the two

most difficult matrices, lodgepole pine leaf litter and high organic clay and analyzing on two

different days, these MDLs must be considered conservative but reflect performance under

routine operating conditions.

4 Guidance Manual on Sampling, Analysis and Data Management, Volume Four, Updated Compendium of

Analytical Methods for Contaminated Sites Section 6.3



Page 38 of 78

Table 5. Method Detection Limit of the Acetone-HPLC and DCM-HPLC Method

µg/g Elemental Sulphur

Acetone Extract Day 1 Day 2 Average

Lodgepole Pine 6.4 5.4 6.8

High Organic Clay 6.2 9.3

DCM Extract Day 1 Day 2 Average

Lodgepole Pine 19 44 33.5

High Organic Clay 35 36

3.2.4 Method Blank

Unspiked samples of Ottawa sand, leaf litters, and clays were analyzed. All values were non-

detect except the high organic clay, which actually does contain 8 µg/g S8. The method

contributes no measurable background.

3.2.5 Precision and Accuracy, Robustness

Low, mid and high level spiked replicates of lodgepole pine leaf litter and high organic clay

extracted with acetone and DCM were analyzed on two separate days. Low level spikes were

prepared by spiking 2 g aliquots of soil using a concentrated solution of S8 in DCM. The spiked

samples were allowed to evaporate overnight in a fume hood prior to extraction. High level

spikes were prepared by weighing the appropriate amount of S8 into a vial containing 2 g of

sample. The soil-S8 mixtures were homogenized by tumbling prior to extraction. Both the

sample preparation and analysis involved two analysts to demonstrate robustness. All data are

included in the appended Master Data Table. Data are summarized in Table 6.

All recoveries are well within acceptance limits. The low level acetone spike of 20 µg/g is only 3x

MDL. Maxxam corporate criteria at this level are: accuracy ± 50%, precision ± 20%. The high

level recoveries (averaging 99%) validate the extraction efficiency at high concentrations of S8.

Overall average recovery is 99.1% with an RSD of 5.3%.



Page 39 of 78

Table 6. Average Percent Recovery and Relative Standard Deviation (RSD) of the Acetone-

HPLC and DCM-HPLC Methods

Sample Spike level

(µg/g) Average Recovery

RSD Sample Size (n)

Acetone Extract

Lodgepole Pine 20 118% 9.2% 8

High Organic Clay 20 113% 11.5% 8



Lodgepole Pine 1,000 84% 1.4% 6

High Organic Clay 1,000 90% 0.8% 6



DCM Extract



Lodgepole Pine 10,000 101% 6.6% 6




Overall Average 99.1% 5.3%

3.2.6 Selectivity

Selectivity was discussed in detail in Section 3.1. The S8 peak is separated from interference

peaks. The chromatograms of high matrix samples show non-detect results at the S8 elution

time. Extraction efficiencies are good. In short, we have found nothing to indicate that the

method will generate either false positives or false negatives.

3.2.7 Second Source Verification

A standard obtained from a second source was analyzed to verify the purity of the primary

standard. Eight replicates prepared at 20 µg/mL were analyzed at regular intervals throughout a



Page 40 of 78

100-sample run. The data are excellent: 98% recovery with an RSD of 3.5%. Recoveries are

tabulated in Table 7.

Table 7. Second Source Verification Results

Replicate Spike Recovery (µg/mL)

ICV-1 19.14

ICV-2 18.53

ICV-3 19.27

ICV-4 29.08*

ICV-5 19.47

ICV-6 20.78

ICV-7 19.87

ICV-8 19.55

Average

(excluding outlier)

19.52

Theoretical 20

% Recovery 98%

% RSD

(excluding outlier)

3.5%

*Outlier. Peak badly tailed. Runs before and after were normal.

3.2.8 Certified Reference Material (CRM)

After an extensive search, a CRM was found. None of the standard suppliers of CRM for

environmental analysis had a soil CRM with elemental sulphur. We identified a CRM for the

mining industry, a mine-tailing sample, Canadian Certified Reference Materials Project, Sulphide

Ore Mill Tailings Reference Materials, RTS-15. The matrix is essentially finely ground rock. The

Certificate of Analysis is provided in Appendix 2. The value for S8 composition given, 0.50% ±

0.16%, is informational, not certified or provisional, but this was the only suitable matrix we

could identify.

The CRM was used to verify the accuracy of the entire process. Neither the sample preparation

technician nor the analyst knew the concentration of S8 in the CRM. CRM results are tabulated

in Table 8.

5 Canmet, 555 Booth Street, Ottawa, ON, K1A 0G1



Page 41 of 78

Table 8. Sulphur Recovery in Certified Reference Material

Replicate

Measured Concentration (µg/g)

rep 1 3,237

rep 2 3,361

rep 3 3,060

rep 4 3,075

rep 5 3,211

Average 3,189

Standard Deviation 125

%RSD 3.9%

Average in % 0.32% ± 0.01%

The Certificate of Analysis for RTS-1 provides only an informational value for elemental sulphur:

0.50% ± 0.16%. Our result at 0.32% ± 0.01% is just below the lower end of the range, but since it

is an informational value, it is acceptable at this time.

The CRM has been included in our round robin. There would be at least four different methods

used. It will be interesting to see if the results of our round robin for this material will be

sufficient for Natural Resources Canada to update their information on RTS-1 to a certified

value.

4 Summary

The method has passed all validation criteria. The completed SOP is appended as Attachment A.

A round robin project on this SOP and other related analytical methods for elemental sulphur

was conducted with a variety of sample matrices and levels of spikes. The detailed approaches

and results are provided as Attachment B.



Page 42 of 78

5 References

Azarova, I.N.; Gorshkov, A.G., Grachev, M.A.; Korzhova, E.N.; Smagunova, A.N., “Determination

of Elemental Sulfur in Bottom Sediments Using High-Performance Liquid

Chromatography, Journal of Analytical Chemistry, Vol. 56, No. 10, 2001, pp. 929–933.

Maynard, D.G. and Addison, P.A., “Extraction and Colorimetric Determination of Elemental

Sulfur in Organic Horizons of Forest Soils”, Can. J. Soil Sc., 65, 811-813.

Tebbe, F.N.; Wasserman, E.; Peet, W.G.; Vatvars, A.; Hayman, AC., “Composition of Elemental

Sulfur in Solution: Equilibrium of S6, S7 and S8 at Ambient Temperatures”, J. Am. Chem.

Soc. 1982, 104, 4971-4972.

Watkinson, J.H.; Lee, A; Lauren, D.R.; “Measurement of Elemental Sulfur in Soil and Sediments:

Field Sampling, Sample Storage, Pretreatment, Extraction and Analysis by High-

performance Liquid Chromatography, Aust. J. Soil Res. 1987, 25, 167-178.

6 Appendices

Appendix 1: Master Data Table

Appendix 2: Certificate of Analysis

7 Attachments

Attachment A: Standard Operating Procedure for the Analysis of Elemental Sulphur

in Alberta Soils by High Performance Liquid Chromatography

Attachment B: Elemental Sulphur Methods Round Robin



Page 43 of 78

8 Acknowledgements

This work was supported by funding from the Land Monitoring Team of ESRD. We also wish to

acknowledge the ongoing support and input from Dr. Z. Chi Chen, Environmental Soil Specialist

and the Project Manager, Land Policy Branch, of the Alberta Environment and Parks; and

contributions from Bonnie Leung, Analytical Chemist, Science Division, Alberta Environmental

Monitoring, Evaluation & Reporting Agency; and Catherine Evans, Risk Assessment Specialist,

Closure and Liability Branch, Alberta Energy Regulator.

This report was completed by the following:

Barry Loescher, PhD, P Chem,

Quality Systems Specialist

Maxxam Analytics

Heather Lord, PhD

Manager, Environmental Research & Development

Maxxam Analytics

Dina Tleugabulova, PhD

Scientific Specialist, Edmonton Environmental

Maxxam Analytics



Page 44 of 78

Appendix 1 Master Data Table

Method Blank

Sample # Sample nameExtraction

solvent

S expected

extract

conc. (mg/L)

S conc.

(mg/L)

2013/03/

04

S conc.

(mg/L)

2013/03/04

with blank

correction

Recovery

%

S conc.

(mg/L)

2013/03/

05

S conc.

(mg/L)

2013/03/05

with blank

correction

Recovery

%

7-1 Ottawa sand Acetone - ND ND NA ND ND NA

7-2 Ottawa sand Acetone - ND ND NA ND ND NA

8 Non-spiked lodgepole Acetone - ND NA NA ND NA NA

9 Non-spiked clay/org 9% Acetone - 0.86 NA NA 0.81 NA NA Average (mg/L) 0.84

ND = Not detected

NA = Not applicable



Page 45 of 78

Precision and Accuracy, MDL

Acetone


solvent

S expected

extract

conc. (mg/L)

S conc.

(mg/L)

Rep 1

S conc.

(mg/L)

Rep 1 with

blank

correction

Recovery %

S conc.

(mg/L)

Rep 2

S conc.

(mg/L)

Rep 2 with

blank

correction

Recovery %

Average S8

conc. in

extract

(mg/L)

Average S8

conc. In soil

(mg/kg)

Average

Recovery

Edmonton Extracts

1-1 Lodgepole Acetone 2 2.18 2.18 109 2.31 2.31 116 2.25 22.5 112.3% Precision

1-2 Lodgepole Acetone 2 2.39 2.39 120 2.48 2.48 124 2.44 24.4 121.8% 9.2%

1-3 Lodgepole Acetone 2 1.96 1.96 98 2.36 2.36 118 2.16 21.6 108.0% Accuracy


1-5 Lodgepole Acetone 2 2.51 2.51 126 2.59 2.59 130 2.55 25.5 127.5% Count

1-6 Lodgepole Acetone 2 1.93 1.93 97 2.20 2.20 110 2.07 20.7 103.3% 8

1-7 Lodgepole Acetone 2 2.18 2.18 109 2.64 2.64 132 2.41 24.1 120.5% Avg. Recovery %


3-1 clay/org 9% Acetone 2 3.36 3.36 168 3.43 3.43 171 3.39 33.9 169.7% Precision

3-2 clay/org 9% Acetone 2 2.77 2.77 139 2.91 2.91 146 2.84 28.4 142.1% 11.5%

3-3 clay/org 9% Acetone 2 3.18 3.18 159 2.88 2.88 144 3.03 30.3 151.5% Accuracy

3-4 clay/org 9% Acetone 2 3.05 3.05 153 2.93 2.93 146 2.99 29.9 149.5% 54.5%

3-5 clay/org 9% Acetone 2 3.17 3.17 159 3.45 3.45 172 3.31 33.1 165.4% Count

3-6 clay/org 9% Acetone 2 3.11 3.11 156 3.62 3.62 181 3.37 33.7 168.3% 8

3-7 clay/org 9% Acetone 2 3.02 3.02 151 2.88 2.88 144 2.95 29.5 147.5% Avg. Recovery %

3-8 clay/org 9% Acetone 2 2.75 2.75 138 2.94 2.94 147 2.84 28.4 142.2% 154.5%





2-5 Lodgepole Acetone 20 21.40 21.40 107 24.49 24.49 122 22.95 229.5 114.7% Count

2-6 Lodgepole Acetone 20 19.50 19.50 98 24.70 24.70 123 22.10 221.0 110.5% 8

2-7 Lodgepole Acetone 20 20.48 20.48 102 19.88 19.88 99 20.18 201.8 100.9% Avg. Recovery %



4-2 clay/org 9% Acetone 20 23.39 23.39 117 19.63 19.63 98 21.51 215.1 107.5% 3.2%


4-4 clay/org 9% Acetone 20 21.33 21.33 107 22.38 22.38 112 21.86 218.6 109.3% 9.6%

4-5 clay/org 9% Acetone 20 20.65 20.65 103 23.04 23.04 115 21.85 218.5 109.2% Count

4-6 clay/org 9% Acetone 20 21.20 21.20 106 24.74 24.74 124 22.97 229.7 114.9% 8

4-7 clay/org 9% Acetone 20 21.12 21.12 106 22.28 22.28 111 21.70 217.0 108.5% Avg. Recovery %

4-8 clay/org 9% Acetone 20 23.33 23.33 117 19.09 19.09 95 21.21 212.1 106.0% 109.6%

04-Mar 05-Mar



Page 46 of 78


Acetone


solvent

S expected

extract

conc. (mg/L)

S conc.

(mg/L)

Rep 1

S conc.

(mg/L)

Rep 1 with

blank

correction

Recovery

%

S conc.

(mg/L)

Rep 2

S conc.

(mg/L)

Rep 2 with

blank

correction

Recovery

%

Average S8

conc. in

extract

(mg/L)

Average S8

conc. In soil

(mg/kg)

Average

Recovery

Mississauga Extracts





2-5 Lodgepole Acetone 100 87.22 87.22 87 84.35 84.35 84 85.79 857.9 85.8% Avg. Recovery % Count

2-6 Lodgepole Acetone 100 86.44 86.44 86 83.96 83.96 84 85.20 852.0 85.2% 84.3% 6


1-2 clay/org 9% Acetone 100 91.69 91.69 92 88.74 88.74 89 90.21 902.1 90.2% 0.8%


1-4 clay/org 9% Acetone 100 90.05 90.05 90 91.46 91.46 91 90.76 907.6 90.8% 9.3%

1-5 clay/org 9% Acetone 100 91.46 91.46 91 90.22 90.22 90 90.84 908.4 90.8% Avg. Recovery % Count

1-6 clay/org 9% Acetone 100 90.13 90.13 90 88.78 88.78 89 89.45 894.5 89.5% 90.7% 6

LP1-High Lev. Lodgepole Acetone 360 357.58 357.58 99 390.4255 390.43 108 374.00 3740.0 103.9% Precision

LP2-High Lev. Lodgepole Acetone 360 340.50 340.50 95 351.1404 351.14 98 345.82 3458.2 96.1% 3.4%

LP3-High Lev. Lodgepole Acetone 360 343.37 343.37 95 353.2575 353.26 98 348.31 3483.1 96.8% Accuracy

LP4-High Lev. Lodgepole Acetone 360 341.24 341.24 95 368.4424 368.44 102 354.84 3548.4 98.6% 2.3%

LP5-High Lev. Lodgepole Acetone 360 329.81 329.81 92 368.469 368.47 102 349.14 3491.4 97.0% Avg. Recovery % Count

LP6-High Lev. Lodgepole Acetone 360 330.92 330.92 92 344.8833 344.88 96 337.90 3379.0 93.9% 97.7% 6

HOC1-High Lev. clay/org 9% Acetone 360 366.91 366.91 102 394.95 394.95 110 380.93 3809.3 105.8% Precision

HOC2-High Lev. clay/org 9% Acetone 360 396.27 396.27 110 379.21 379.21 105 387.74 3877.4 107.7% 3.6%

HOC3-High Lev. clay/org 9% Acetone 360 395.70 395.70 110 393.91 393.91 109 394.80 3948.0 109.7% Accuracy

HOC4-High Lev. clay/org 9% Acetone 360 393.26 393.26 109 400.07 400.07 111 396.67 3966.7 110.2% 6.4%

HOC5-High Lev. clay/org 9% Acetone 360 365.09 365.09 101 384.41 384.41 107 374.75 3747.5 104.1% Avg. Recovery % Count

HOC6-High Lev. clay/org 9% Acetone 360 326.65 326.65 91 399.51 399.51 111 363.08 3630.8 100.9% 106.4% 6

26-Mar 27-Mar

15-Mar 18-Mar



Page 47 of 78


DCM


solvent

S expected

extract

conc. (mg/L)

S conc.

(mg/L)

Rep 1

S conc.

(mg/L)

Rep 1 with

blank

correction

Recovery

%

S conc.

(mg/L)

Rep 2

S conc.

(mg/L)

Rep 2 with

blank

correction

Recovery

%

Average S8

conc. in

extract

(mg/L)

Average S8

conc. In soil

(mg/kg)

Average

Recovery


5-1 Lodgepole DCM 20 17.75 17.75 89 18.47 18.47 92 18.11 181.1 90.6% Precision

5-2 Lodgepole DCM 20 19.38 19.38 97 20.78 20.78 104 20.08 200.8 100.4% 4.5%

5-3 Lodgepole DCM 20 18.97 18.97 95 18.13 18.13 91 18.55 185.5 92.8% Accuracy

5-4 Lodgepole DCM 20 18.60 18.60 93 16.23 16.23 81 17.42 174.2 87.1% 6.5%

5-5 Lodgepole DCM 20 18.35 18.35 92 17.20 17.20 86 17.77 177.7 88.9% Avg. Recovery %

5-6 Lodgepole DCM 20 18.80 18.80 94 20.20 20.20 101 19.50 195.0 97.5% 93.5%

5-7 Lodgepole DCM 20 19.09 19.09 95 18.99 18.99 95 19.04 190.4 95.2% Count

5-8 Lodgepole DCM 20 19.83 19.83 99 18.43 18.43 92 19.13 191.3 95.6% 8

3-1 clay/org 9% DCM 20 20.42 20.42 102 19.26 19.26 96 19.84 198.4 99.2% Precision

3-2 clay/org 9% DCM 20 19.06 19.06 95 18.63 18.63 93 18.84 188.4 94.2% 4.1%

3-3 clay/org 9% DCM 20 18.10 18.10 90 17.76 17.76 89 17.93 179.3 89.6% Accuracy

3-4 clay/org 9% DCM 20 21.02 21.02 105 18.49 18.49 92 19.76 197.6 98.8% 6.6%

3-5 clay/org 9% DCM 20 18.25 18.25 91 18.22 18.22 91 18.23 182.3 91.2% Avg. Recovery %

3-6 clay/org 9% DCM 20 20.18 20.18 101 16.21 16.21 81 18.20 182.0 91.0% 93.4%

3-7 clay/org 9% DCM 20 18.54 18.54 93 20.05 20.05 100 19.30 193.0 96.5% Count

3-8 clay/org 9% DCM 20 17.94 17.94 90 16.90 16.90 84 17.42 174.2 87.1% 9

3-9 clay/org 9% DCM 20 20.07 20.07 100 17.30 17.30 87 18.69 186.9 93.4%

15-Mar 18-Mar



Page 48 of 78


DCM


solvent

S expected

extract

conc. (mg/L)

S conc.

(mg/L)

Rep 1

S conc.

(mg/L)

Rep 1 with

blank

correction

Recovery

%

S conc.

(mg/L)

Rep 2

S conc.

(mg/L)

Rep 2 with

blank

correction

Recovery

%

Average S8

conc. in

extract

(mg/L)

Average S8

conc. In soil

(mg/kg)

Average

Recovery


LP1-Mid Lev. Lodgepole DCM 1000 984.694 984.69 98 1059.84 1059.84 106 1022.27 10222.7 102.2% Precision

LP2-Mid Lev. Lodgepole DCM 1000 888.3687 888.37 89 1128.82 1128.82 113 1008.59 10085.9 100.9% 6.6%

LP3-Mid Lev. Lodgepole DCM 1000 1111.773 1111.77 111 1119.56 1119.56 112 1115.67 11156.7 111.6% Accuracy

LP4-Mid Lev. Lodgepole DCM 1000 836.8425 836.84 84 995.80 995.80 100 916.32 9163.2 91.6% 1.1%

LP5-Mid Lev. Lodgepole DCM 1000 887.3657 887.37 89 1056.96 1056.96 106 972.16 9721.6 97.2% Avg. Recovery % Count

LP6-Mid Lev. Lodgepole DCM 1000 996.9422 996.94 100 1062.91 1062.91 106 1029.93 10299.3 103.0% 101.1% 6

HOC1-Mid Lev. clay/org 9% DCM 1000 878.09 878.09 88 802.15 802.15 80 840.12 8401.2 84.0% Precision

HOC2-Mid Lev. clay/org 9% DCM 1000 848.67 848.67 85 1098.33 1098.33 110 973.50 9735.0 97.3% 7.1%

HOC3-Mid Lev. clay/org 9% DCM 1000 887.09 887.09 89 1106.84 1106.84 111 996.96 9969.6 99.7% Accuracy

HOC4-Mid Lev. clay/org 9% DCM 1000 914.24 914.24 91 1092.23 1092.23 109 1003.23 10032.3 100.3% 2.2%

HOC5-High Lev. clay/org 9% DCM 1000 970.06 970.06 97 1051.26 1051.26 105 1010.66 10106.6 101.1% Avg. Recovery % Count

HOC6-Mid Lev. clay/org 9% DCM 1000 1047.59 1047.59 105 1036.71 1036.71 104 1042.15 10421.5 104.2% 97.8% 6

LP1-High Lev. Lodgepole DCM 3600 3349.48 3349.48 93 3493.96 3493.96 97 3421.72 34217.2 95.0% Precision

LP2-High Lev. Lodgepole DCM 3600 3612.84 3612.84 100 2017.09 2017.09 56 * - * 4.3%

LP3-High Lev. Lodgepole DCM 3600 3541.80 3541.80 98 3582.81 3582.81 100 3562.30 35623.0 99.0% Accuracy

LP4-High Lev. Lodgepole DCM 3600 3578.09 3578.09 99 3283.38 3283.38 91 3430.74 34307.4 95.3% 2.3%

LP5-High Lev. Lodgepole DCM 3600 3929.76 3929.76 109 3606.04 3606.04 100 3767.90 37679.0 104.7% Avg. Recovery % Count

LP6-High Lev. Lodgepole DCM 3600 3650.82 3650.82 101 3160.41 3160.41 88 3405.62 34056.2 94.6% 97.7% 5

HOC1-High Lev. clay/org 9% DCM 3600 3595.72 3595.72 100 3468.36 3468.36 96 3532.04 35320.4 98.1% Precision

HOC2-High Lev. clay/org 9% DCM 3600 3011.81 3011.81 84 3044.31 3044.31 85 3028.06 30280.6 84.1% 5.5%

HOC3-High Lev. clay/org 9% DCM 3600 3390.46 3390.46 94 3590.06 3590.06 100 3490.26 34902.6 97.0% Accuracy

HOC4-High Lev. clay/org 9% DCM 3600 3491.15 3491.15 97 3315.71 3315.71 92 3403.43 34034.3 94.5% 7.3%

HOC5-High Lev. clay/org 9% DCM 3600 3174.57 3174.57 88 3161.50 3161.50 88 3168.03 31680.3 88.0% Avg. Recovery % Count

HOC6-High Lev. clay/org 9% DCM 3600 3475.32 3475.32 97 3308.51 3308.51 92 3391.91 33919.1 94.2% 92.7% 6

*Outlier - removed from calculation

26-Mar 27-Mar

26-Mar 27-Mar



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Appendix 2 Certificate of Analysis



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Attachment A. Standard Operating Procedure for the Analysis of Elemental Sulphur in Alberta Soils by High Performance Liquid

Chromatography

Developed by Maxxam Analytics for Alberta Environment and Parks

June, 2015



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Attachment A - Table Of Contents

1 Introduction ...................................................................................................................... 54 1.1 Scientific Principle ................................................................................................ 54

2 Scope ................................................................................................................................. 54 2.1 Applicability .......................................................................................................... 54

3 Definitions and Acronyms ................................................................................................ 55 4 Reference Method ............................................................................................................ 56

4.1 Primary Reference ................................................................................................ 56 4.2 Secondary Reference ............................................................................................ 56 4.3 Deviations from the Primary Reference ............................................................... 56

5 Regulatory Criteria ............................................................................................................ 56 6 Safety And Disposal .......................................................................................................... 57 7 Sample Handling, Preservation And Hold Time ............................................................... 58 8 Interferences ..................................................................................................................... 58 9 Detection Limit ................................................................................................................. 59

9.1 Method Detection Limit (MDL) ............................................................................ 59 9.2 Reporting Limit (RL) .............................................................................................. 59 9.3 Measurement of Uncertainty ............................................................................... 59 9.4 Accuracy and Precision ......................................................................................... 59

10 Glassware Cleaning ........................................................................................................... 59 11 Apparatus And Materials ................................................................................................. 60

11.1 Materials............................................................................................................... 60 11.2 Apparatus ............................................................................................................. 60

12 Reagents and Standard Preparation ................................................................................ 61 12.1 Reagents ............................................................................................................... 61 12.2 Calibration Standards ........................................................................................... 62 12.3 Quality Control Standards .................................................................................... 62

13 Sample Preparation .......................................................................................................... 63 14 Analytical Determination ................................................................................................. 64

14.1 Instrument Setup.................................................................................................. 64 14.2 Column Performance Check ................................................................................. 64 14.3 Instrument Calibration ......................................................................................... 65

14.3.1 Initial Calibration ..................................................................................... 65 14.3.2 Calibration Acceptance Criteria............................................................... 65 14.3.3 Initial Calibration Verification (ICV) and

Continuing Calibration Verification (CCV) ............................................... 66 14.3.4 Run Format .............................................................................................. 67

15 Quality Control Requirements ......................................................................................... 68 16 Data Interpretation, Calculations And Data Reporting ................................................... 69

16.1 Calculations .......................................................................................................... 69 16.2 Analyte Identification ........................................................................................... 70 16.3 Significant Figures ................................................................................................ 70

17 Column Maintenance ....................................................................................................... 70 17.1 Column Cleaning Protocol .................................................................................... 70



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1 Introduction

Elemental sulphur is one of the regulated substances in the Province of Alberta and analytical

methods for its determination need to be further developed for monitoring and mitigating its

impact on soils. Maxxam Analytics International Corporation was previously contracted by the

former Department of Alberta Environment and Sustainable Resource Development (ESRD) to

develop a High Performance Liquid Chromatography (HPLC) analytical method for quantitation

of elemental sulphur in soils. This Standard Operating Procedure (SOP) is provided to ensure

consistency in methodology and data quality objectives, following completion of the round

robin by participating laboratories.

1.1 Scientific Principle

Sulphur is one of the non-metal elements that is abundantly available in the elemental form. It is

an odourless, brittle, yellow solid that is easily oxidized or reduced, depending on its

environment. Sulphur in the elemental form is used directly in the manufacture of pulp and

paper, carbon disulfide, fertilizers, and rubber and other elastomers. It is used in industry in the

form of sulphuric acid. Sulphur occurs naturally near volcanoes and hot springs as well as in

natural gas and petroleum crudes. Elemental sulphur is slowly converted to sulfate in soil by the

action of autotrophic bacteria, the most important of which belong to the genus Thiobacillus.

Variability in elemental sulphur oxidation rates among soils is reported6 to be related to the

differences in the number of Thiobacillus. The oxidized sulphur slowly leaches from the soil as

sulfate.

Elemental sulphur (S8) is insoluble in water and only slightly soluble in organic solvents such as

ether, petroleum ether, toluene, chloroform, and alcohol.

In this method, after drying and grinding to < 2 mm, soil samples are extracted with either

acetone or dichloromethane (DCM). These solvents effectively extract all forms of elemental

sulphur from the soil matrix up to concentrations limited by the solubility of S8 in the solvent.

The extract is separated from the soil residue and analyzed by High Performance Liquid

Chromatography (HPLC) using a methanol-water mobile phase and UV detection at 220 nm.

2 Scope

2.1 Applicability

This method is suitable for determination of elemental sulphur in soils at concentrations from

___________________ 6 Havlin, J.L., Beaton, J.D., Tisdale S.L. and Nelson, W.L., 1999. Soil Fertility and Fertilizers

– An Introduction to Nutrient Management. 6th ed., Prentice Hall Inc., New Jersey.



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10 to 40,000 mg/kg. The range can be extended by dilution and/or extraction at a higher solvent

to soil ratio.

3 Definitions and Acronyms

Continuing Calibration Verification (CCV): Usually the midpoint standard of the calibration

curve, analyzed to verify the applicability of the calibration curve.

Initial Calibration Blank (ICB): Continuing Calibration Blank (CCB): Aliquots of pure solvent,

analyzed to assess baseline stability.

Initial Calibration Verification (ICV): A standard prepared from a secondary source analyzed to

verify the calibration standards.

Laboratory Control Sample (LCS) (also referred to as a Blank Spike): A sample of known

concentration used as a basis for comparison with test samples that undergoes sample

processing identical to that carried out for test samples.

Laboratory Duplicate Sample: One of two sample aliquots obtained from the same sample

container and carried through the entire analytical process. Also referred to as a split sample.

LIMS: Laboratory Information Management System.

Matrix Spike: A second aliquot of sample spiked with a known amount of the analyte, used to

assess matrix interference.

Method Blank: A blank sample that undergoes sample processing identical to that carried out

for test samples. Method blank results are used to assess contamination from the laboratory

environment and reagents.

Relative Percent Difference (RPD): The absolute difference between two results expressed as a

percentage of the average result:

100221

21

xx

xxRPD

Standard Deviation (SD): A measure of the dispersion or imprecision of a sample or population

distribution expressed as the positive square root of the variance, with the same unit of

measurement as the mean.

Relative Standard Deviation (RSD): The standard deviation of an array X divided by the average

of the array, times 100. Expressed as a percentage:

RSD = [SD(1-X) / average(1 – X)] x 100



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4 Reference Method

4.1 Primary Reference

“Determination of Elemental Sulphur in Bottom Sediments Using High-Performance Liquid

Chromatography”, I. N. Azarova, et al., Journal of Analytical Chemistry, Vol. 56, No. 10, 2001, pp.

929–933.

4.2 Secondary Reference

“Measurement of Elemental Sulphur in Soil and Sediments: Field Sampling, Sample Storage,

Pretreatment, Extraction and Analysis by High-performance Liquid Chromatography”, J.H.

Watkinson et al., Aust. J. Soil Res. 1987, 25, 167-178.

4.3 Deviations from the Primary Reference

Deviations have been demonstrated fit for purpose by method validation and performance.

Reference Method SOP Justification

5 g sample extracted with 3 x 15 mL portions of acetone in an ultrasonic bath

2 g sample extracted with 20 mL acetone, sonicated for 30 min. followed by 1 hr. tumbling

Excellent recoveries up to 400 mg/L

5 g sample extracted with 3 x 15 mL portions of acetone in an ultrasonic bath

2 g samples also extracted with 20 mL DCM

S8 has 10x greater solubility in DCM than in acetone. Allows determination of up to 6% S8 in soil

Acetonitrile-water mobile phase

Methanol-water mobile phase

S8 has greater solubility in methanol allowing a wider linear range than the reference method

Column (2 x 75 mm)

packed with the sorbent Nucleosil 100-5 C18

AkzoNobel Kromasil C18 column (4.6 x 100 mm, 3.5 µm particle size)

Commercially available, higher capacity, provides excellent separation of the S8 peak

5 Regulatory Criteria

500 mg elemental S/kg of soil per Alberta Tier 1 Soil and Water Remediation Guidelines, May 23,

2014, or as amended.



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6 Safety and Disposal

6.1 The use of personal protective equipment, including safety glasses and lab coats is required. It is the responsibility of the analyst to ensure that any additional identified hazard controls are used (e.g. nitrile gloves, splash goggles, fume hoods, respirators, etc.)

6.2 Material safety data sheets for all chemical reagents must be available to personnel using this method. Staff performing this method shall review the associated MSDS sheets for chemicals used in this procedure and ensure they understand the associated hazards and safety controls required to work safely with each chemical.

6.3 Dispose of all samples, extracts and reagents according to local, provincial and federal laws and regulations.

6.4 CAUTION: Samples containing > 30% S8 may spontaneously ignite on grinding. Suspected high S8 samples should be tested using minimal sample aliquots and not ground if ignitability is suspected.



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7 Sample Handling, Preservation and Hold Time

Matrix Container Minimum Amount

Hold Time Preservation

Soil Extract Conical glass tubes with caps

10 mL 14 days Store at 4 ± 2°C

Soil Wide mouth glass jars (recommended*), plastic bags or polyethylene containers of various sizes.

125 mL 14 days** Store at 4 ± 2°C.

Dried and Ground Soil

Glass 10 grams indefinite Store at room temperature

* S8 may tend to adhere electrostatically to plastic surfaces ** Soils should be dried and ground as soon as possible after receipt in order to minimize biological degradation of

S8. Freezing as-received samples may be used to extend hold time.

8 Interferences

8.1 Acetone extracts with S8 concentrations ≥ 400 mg/L and DCM extracts with concentrations ≥ 6,500 mg/L must be re-extracted at a higher solvent:soil ratio. Studies have shown that soils with concentrations that yield extract concentrations above these levels are not completely extracted.

8.2 Co-extracted organics could potentially interfere with the S8 peak. HPLC conditions have been optimized such that the S8 peak elutes after most of these interferences.

8.3 S8 standards contain small amounts of S6 and S7 allotropes. Our studies have shown that these and other impurities are < 2% of the S8, therefore not significant.

8.4 Leaf litter samples may contain pine pitch or other interferences that can clog the guard column relatively quickly. Frequent (daily) guard column replacement will be required if high numbers of such samples are run.



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9 Detection Limit

9.1 Method Detection Limit (MDL)

MDL must be re-determined at a minimum every two years or if a major change is introduced to

the test method. Typical MDLs are 7 mg/kg for an acetone extract and 35 mg/kg for a DCM

extract.

9.2 Reporting Limit (RL)

Acetone extract RL = 10 mg/kg. DCM extract RL = 40 mg/kg. Data less than the RL should be

reported as < (RL) on the Certificate of Analysis. Any applicable sample dilution factors are

applied to the RL values, which are reported accordingly.

9.3 Measurement of Uncertainty

The uncertainty value represents the variability of the result due to both random and systematic

causes, thus providing a level of confidence when making a decision using a result. Bias

estimates less than 5% are not included in this procedure.

9.4 Accuracy and Precision

The accuracy and precision of the method were estimated from replicate analyses of spiked real

sample matrice

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